An asK1 / pdk1 dual-targeting inhibitor and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to an ASK1 / PDK1 dual-targeting inhibitor, a pharmaceutically acceptable salt thereof, an ester thereof and optical isomers thereof. The compound disclosed by the application can be used as an inhibitor targeting ASK1 and PDK1, and can be applied to the treatment and / or prevention of ASK1 / PDK1 dual-targeting mediated fibrosis disease related diseases in medicine. The compound of the application is a compound as shown in general formula I and optical isomers, pharmaceutically acceptable salts and solvates thereof, wherein R1 is R12 is selected from C3-C8 cycloalkyl, methyl, ethyl, C3-C8 straight chain or branched alkyl; R13 is selected from methyl, ethyl or propyl; and R2 is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to ASK1 / PDK1 dual-targeting inhibitors, their pharmaceutically acceptable salts, their esters, and their optical isomers. The compounds of this invention can be used as inhibitors targeting ASK1 and PDK1 in pharmaceuticals for the treatment and / or prevention of ASK1 / PDK1 dual-targeting mediated fibrotic diseases. Background Technology

[0002] Apoptosis signal-regulated kinase 1 (ASK1) is a widely expressed serine / threonine kinase. Knockout or inhibition of ASK1 can effectively prevent inflammation and fibrosis. Under pathological conditions of cellular stress, ASK1 can be activated by factors such as reactive oxygen species (ROS), lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and endoplasmic reticulum (ER) stress, thereby phosphorylating downstream JNKs and p38 MAPKs proteins and affecting signaling pathways such as inflammation, fibrosis, and apoptosis.

[0003] 3-Phosphoinositol-dependent protein kinase 1 (PDK1) is a kinase within the AGC protein family that is highly expressed in various cell types, including endothelial cells and fibroblasts. It phosphorylates its downstream factor AKT, thereby regulating cell proliferation, apoptosis, and differentiation. Studies have shown that PDK1 activity is closely related to fibrosis; inhibiting PDK1 can suppress cell signaling pathways and thus promote fibroblast apoptosis.

[0004] The ASK1 inhibitor GS-4997 has been clinically studied in diabetic nephropathy, alcoholic fatty liver disease, and pulmonary hypertension. However, compared with placebo, GS-4997 monotherapy showed no significant effect in clinical trials. This may be due to the complex interactions between various signaling pathways; inhibiting one pathway alone may affect the effects of others. Therefore, the development of ASK1 inhibitors needs to address the limitations of monotherapy by targeting multiple targets with a single molecule. ASK1 and PDK1 have similar regulatory effects in cells, but studies have shown that there is also a negative feedback effect between PDK1 and ASK1. Patel et al. reported that the combination of a PDK1 inhibitor (BX-795) and an ASK1 inhibitor (GS-4997) can achieve antithrombotic effects.

[0005]

[0006] Invention patents CN110294746, CN110698471, and CN110407806 disclose novel ASK1 inhibitors. To date, these compounds only inhibit ASK1, and there are no research findings on dual-targeting ASK1 / PDK1 inhibitors. Therefore, dual-targeting ASK1 / PDK1 inhibitors represent an important direction in compound research and have significant potential value. Summary of the Invention

[0007] The purpose of this invention is to provide a compound with dual ASK1 / PDK1 inhibitory effects, which provides the possibility for the preparation of drugs for the treatment and / or prevention of fibrotic diseases mediated by dual ASK1 / PDK1 targeting.

[0008] The specific technical solution is as follows:

[0009] This invention claims protection for compounds of general formula (I), their pharmaceutically acceptable salts, esters, and solvates:

[0010]

[0011] Where R1 is R12 is selected from C3-C8 cycloalkyl, methyl, ethyl, C3-C8 straight-chain or branched alkyl; R13 is selected from methyl, ethyl or propyl;

[0012] R2 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0013] Preferably, R12 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, isopropyl, n-propyl, n-butyl, and isobutyl.

[0014] This invention claims protection for compounds of general formula (II), their pharmaceutically acceptable salts, esters, and solvates:

[0015]

[0016] Among them, R3 is R14 is selected from C3-C8 cycloalkyl, methyl, ethyl, C3-C8 straight-chain or branched alkyl;

[0017] R4 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0018] Preferably, R14 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, isopropyl, n-propyl, n-butyl, and isobutyl.

[0019] This invention claims protection for compounds of general formula (III), their pharmaceutically acceptable salts, esters, and solvates:

[0020]

[0021] Where X is C or N;

[0022] R5 is hydrogen, C1-C3 alkyl, or halo-C1-C3 alkyl; R6 is hydrogen, C1-C3 alkyl, halo-C1-C3 alkyl, or substituted benzene ring; R7 is hydrogen or substituted benzene ring; R8 is C3-C8 cycloalkyl, C1-C6 alkyl, or C1-C6 hydroxyalkyl.

[0023] Preferably, R6 is methyl,

[0024] Preferably, when R7 is a substituted benzene ring, the substituent is one or more of methyl, ethyl, propyl, and halogen.

[0025] Preferably, when R7 is a substituted benzene ring, the substituents are methyl and halogen.

[0026] Preferably, when R7 is a substituted benzene ring, the methyl group and the halogen are ortho-substituted on the benzene ring.

[0027] Preferably, R8 is a C3-C6 cycloalkyl, C1-C4 alkyl, or C1-C4 hydroxyalkyl.

[0028] Preferably, R8 is isopropyl, n-propyl, or C3 hydroxyalkyl.

[0029] Preferably, R8 is -CH(CH3)CH2OH.

[0030] This invention claims protection for compounds of general formula (IV), their pharmaceutically acceptable salts, esters, and solvates:

[0031]

[0032] Where Y is C or N;

[0033] R9 is a five-membered heterocycle, a substituted five-membered heterocycle, a six-membered heterocycle, or a substituted six-membered heterocycle; the substituents on the substituted five-membered heterocycle and the substituted six-membered heterocycle are selected from C3-C8 cycloalkyl, C1-C6 alkyl, and C1-C6 haloalkyl.

[0034] R10 is hydrogen, a substituted benzene ring;

[0035] R11 is a C3-C8 cycloalkyl, C1-C6 alkyl, or C1-C6 hydroxyalkyl.

[0036] Preferably, R9 is pyrrole, substituted pyrrole, pyrazole, substituted pyrazole, imidazole, substituted imidazole, pyridine, substituted pyridine, pyrimidine, or substituted pyrimidine; the substituents on the substituted pyrrole, substituted pyrazole, substituted imidazole, substituted pyridine, and substituted pyrimidine are selected from C3-C8 cycloalkyl, C1-C6 alkyl, and C1-C6 haloalkyl.

[0037] Preferably, the substituents on the substituted pyrrole, substituted pyrazole, substituted imidazole, substituted pyridine, and substituted pyrimidine are selected from C3-C6 cycloalkyl, C1-C4 alkyl, and C1-C4 haloalkyl.

[0038] Preferably, the substituents on the substituted pyrrole, substituted pyrazole, substituted imidazole, substituted pyridine, and substituted pyrimidine are selected from isopropyl, n-propyl, n-butyl, and isobutyl.

[0039] Preferably, R9 is

[0040] Preferred, R 10 When the benzene ring is substituted, the substituent is one or more of methyl, ethyl, propyl, and halogen.

[0041] Preferred, R 10 When the benzene ring is substituted, the substituents are methyl and halogen.

[0042] Preferred, R 10 When the benzene ring is substituted, the methyl group and halogen are ortho-substituted on the benzene ring.

[0043] Preferred, R 11 It is C3-C6 cycloalkyl, C1-C4 alkyl, or C1-C4 hydroxyalkyl.

[0044] Preferred, R 11 It is isopropyl, n-propyl, or C3 hydroxyalkyl.

[0045] Preferred, R 11 It is -CH(CH3)CH2OH.

[0046] Preferably, the present invention claims protection for the following compounds, their optical isomers, pharmaceutically acceptable salts, and solvates, but these compounds do not imply any limitation on the present invention:

[0047] N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 01)

[0048] N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 02)

[0049] N-(3-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazole[3,4-b]pyridine-5-carboxamide (Compound 03)

[0050] N-(6-(4-cyclobutyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (compound 04)

[0051] N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 05)

[0052] N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(2-fluorobenzyl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 06)

[0053] N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(4-methoxybenzyl)-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 07)

[0054] 5-(1-Cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-Cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 08)

[0055] N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 09)

[0056] 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 10)

[0057] N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-6-oxo-1,6-dihydro-[3,3'-bipyridine]-5-carboxamide (compound 11)

[0058] N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (compound 12)

[0059] N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (compound 13)

[0060] 5-(1-Cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-Cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 14)

[0061] 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 15)

[0062] 6-(1-Cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-Cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-4-carboxamide (Compound 16)

[0063] 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indazole-3-carboxamide (Compound 17)

[0064] N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1-isopropyl-1H-pyrazol-4-yl)quinoline-4-carboxamide (compound 18)

[0065] N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(6-methylpyridin-3-yl)quinoline-3-carboxamide (Compound 19)

[0066] N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazole-4-yl)-1H-indazole-3-carboxamide (Compound 20)

[0067] 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indazole-3-carboxamide (Compound 21)

[0068] 5-(1-Cyclopropyl-1H-pyrazole-4-yl)-1-(2-fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 22)

[0069] 1-(2-Fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 23)

[0070] The "pharmaceutically acceptable salt" mentioned in this invention refers to the addition salts and solvates of pharmaceutically acceptable acids and bases.

[0071] The term "ester" as used in this invention refers to a pharmaceutically acceptable ester, particularly esters that are hydrolyzed in vivo and include esters that readily decompose in the human body, leaving behind a parent compound (compounds of general formulas (I) and (II)) or its salt.

[0072] This invention also claims methods for preparing compounds represented by the above general formulas (I-IV), all of which are prepared by methods well known to those skilled in the art of organic chemistry or are commercially available, as described in these routes. All final derivatives of this invention are prepared by methods described in these routes or by similar methods well known to those skilled in the art of organic chemistry. All variables used in these routes are defined below or as defined in the claims.

[0073] According to route 1, compounds of general formula (Ⅰ) can be synthesized.

[0074]

[0075] Step 1: Intermediate 1 is synthesized from an aromatic heterocyclic carboxylic acid via an acylation reaction to obtain intermediate 2.

[0076] Step 2: Intermediate 2 reacts with aromatic heterocyclic borate esters via the Suzuki reaction to obtain compound of general formula (Ⅰ).

[0077] R1 and R2 are defined as before.

[0078] The synthesis of intermediate 1 is as shown in route 2:

[0079]

[0080] Step 1: Compound 1-1 reacts with hydrazine hydrate to produce 1-2

[0081] Step 2: React 1-2 with alkylamines to generate intermediate 1

[0082] Wherein, R2 is isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, halogenated C3-6 cycloalkyl, cyano-substituted C1-4 alkyl, or hydroxy-substituted C1-4 alkyl.

[0083] According to route 3, compounds of general formula (II) can be synthesized.

[0084]

[0085] The synthesis methods for intermediates 3 and 4 are the same as those for route 1.

[0086] Compounds of general formula (III) can be synthesized via route 4.

[0087]

[0088] The synthesis method of intermediate 5 of compound (III) is the same as that of route 2. According to the synthesis route 5, intermediate 6 is obtained. First, 1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-ol is reacted by Vilsmeier-Haack-Arnold to obtain compound 6-2. After reduction and cyclization, compound 6-5 is obtained. Finally, intermediate 6 is obtained by substitution and decarboxylation.

[0089]

[0090] R5 is selected from CH3 and CF3; R6 is selected from CH3. R7 is selected from hydrogen, CH3, CH3CH2, etc.

[0091] Compounds of general formula (Ⅳ) can be synthesized via route 6.

[0092]

[0093] Intermediate 7 and Intermediate 8 are synthesized using the same method as in Route 1. R9, R 10 R 11 The definition is the same as before.

[0094] This invention also claims protection for the use of compounds represented by the above general formulas (I-IV) in medicaments for the treatment and / or prevention of ASK1 / PDK1 dual-targeting mediated diseases.

[0095] Preferably, the ASK1 / PDK1 dual-target mediated disease is fibrosis.

[0096] The present invention also provides a pharmaceutical composition comprising the aforementioned compound, a pharmaceutically acceptable salt thereof, an ester thereof, or a stereoisomer thereof.

[0097] In some specific embodiments of the present invention, the pharmaceutical composition further comprises one or more pharmaceutical carriers and / or diluents.

[0098] The present invention also provides a pharmaceutical formulation comprising the aforementioned compound, a pharmaceutically acceptable salt, ester or stereoisomer thereof, and one or more pharmaceutical carriers and / or diluents; the pharmaceutical formulation being any clinically or pharmaceutically acceptable dosage form.

[0099] In some embodiments of the present invention, the above-described pharmaceutical preparations can be administered to patients or subjects requiring such treatment via oral, parenteral, rectal, or pulmonary administration. For oral administration, the pharmaceutical composition can be formulated into oral preparations, such as conventional oral solid dosage forms like tablets, capsules, pills, granules, etc.; or into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulating oral preparations, suitable fillers, binders, disintegrants, lubricants, etc., can be added. For parenteral administration, the above-described pharmaceutical preparations can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating injections, conventional methods in the existing pharmaceutical field can be used. When preparing injections, excipients may not be added, or suitable excipients may be added depending on the properties of the drug. For rectal administration, the pharmaceutical composition can be formulated into suppositories, etc. For pulmonary administration, the pharmaceutical composition can be formulated into inhalers or sprays, etc.

[0100] The pharmaceutical carriers and / or diluents used in the pharmaceutical compositions or formulations of the present invention can be any conventional carriers and / or diluents in the field of pharmaceutical formulation. The selection of a specific carrier and / or diluent will depend on the route of administration or the type and state of disease for treating a particular patient. The preparation method of a suitable pharmaceutical composition for a specific route of administration is entirely within the knowledge of those skilled in the art of pharmaceuticals. For example, pharmaceutical carriers and / or diluents may include solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, etc., conventional in the pharmaceutical field. If necessary, flavoring agents, preservatives, and sweeteners, etc., may also be added to the pharmaceutical composition.

[0101] Advantages of this invention:

[0102] 1. The compounds synthesized in this invention have good inhibitory activity against ASK1 / PDK1.

[0103] 2. The compounds synthesized in this invention can inhibit the excessive proliferation of fibrotic cells by inhibiting the ASK1 / PDK1 dual pathway, and can be applied to related drugs. Attached Figure Description

[0104] Figure 1 The results show the effects of the compounds of this invention on the PDK1 pathway.

[0105] Figure 2 The results show the effects of the compounds of this invention on the AKT pathway. Detailed Implementation

[0106] The technical solution of the present invention will be described below with reference to specific embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0107] Representative compounds of this invention are shown in Table 1:

[0108] Table 1 Representative compounds of this invention

[0109]

[0110]

[0111] Example 1

[0112] Synthesis of Compound 1

[0113] 1.1 Preparation of 6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine

[0114]

[0115] Step 1: Dissolve methyl 6-aminopyridinecarboxylate (20 g, 132 mmol) in 250 ml of methanol, add hydrazine hydrate solution (13 g, 260 mmol), and react at 80 °C for 8 h. After the reaction is complete, cool the reaction solution to 0 °C, filter, wash the obtained solid with methanol, and dry to obtain 16 g of the target product, with a yield of 80%.

[0116] Step 2: 6-Aminopyridine carboxylhydrazide (5 g, 26 mmol) and triethyl orthoformate were dissolved in 100 ml of 1,4-dioxane and reacted at 75 °C for 2 hours. Cyclopropylamine (1.87 g, 33 mmol) and acetic acid (1.97 g, 33 mmol) were added sequentially, and the reaction was carried out at 100 °C for 12 hours. After the starting materials had reacted completely, the reaction solution was concentrated, and the residue was subjected to silica gel column chromatography (CH3OH / DCM = 1:30) to obtain 5.16 g of the target compound, with a yield of 78%.

[0117] 1.2 Preparation of N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0118]

[0119] 1-Methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid (500 mg, 1.92 mmol) was dissolved in 10 mL of sulfoxide and reacted at 40 °C for 2 h. The solvent was concentrated, and 3-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine (150 mg, 0.74 mmol) was added and dissolved in 15 mL of dichloromethane. The reaction was carried out at room temperature for 5 h. After the reaction was completed, the reaction solution was concentrated, and the residue was subjected to column chromatography (CH3OH / DCM = 1:30) to give 50 mg of the target compound, with a yield of 19%. 1 H NMR (400MHz, DMSO-d6) δ14.01(s,1H),8.68(s,1H),8.58(s,1H),8.36(d,J=8.2Hz,1H),8.00(t,J=8.0 Hz,1H),7.83(d,J=7.6Hz,1H),3.89(s,3H),3.87–3.81(m,1H),1.22–1.15(m,2H),1.10–1.03(m,2H). 13C NMR(151MHz,DMSO-d6)δ=164.15,152.62,151.86,146.20,145.96,139.87,133 .64,122.82,121.04,118.39,115.71,114.30,102.32,35.01,29.41,8.20.ESI m / z:445.1348[M+H] + Found: 445.1348

[0120] Example 2

[0121] Synthesis of Compound 2

[0122] Preparation of N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0123] Following the method in step 1.1 of Example 1, the intermediate 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine was prepared by cyclization reaction with isopropylamine using the intermediate 6-aminopyridinecarboxylhydrazine as a raw material, with a yield of 80%.

[0124] Following the method in step 1.2 of Example 1, the target compound was prepared by acylation of the intermediate 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine with 1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid, with a yield of 21%. 1 H NMR (400MHz, DMSO-d6) δ14.00(s,1H),12.62(s,1H),8.92(s,1H),8.72(s,1H),8.34(d,J=8.3Hz ,1H),8.05(s,1H),7.86(d,J=7.6Hz,1H),5.44–5.32(m,1H),4.00(s,3H),1.55(d,J=6.7Hz,6H). 13 C NMR (151MHz, DMSO-d6)δ=163.47,158.77,150.07,143.51,142.15,139.85,112.53,111.06,49.36,34.61,34.52,23.37.ESI m / z:447.1505[M+H] + Found: 447.1516

[0125] Example 3

[0126] Synthesis of Compound 3

[0127] Preparation of N-(3-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazole[3,4-b]pyridine-5-carboxamide

[0128] 3.1 Preparation of 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)aniline

[0129]

[0130] Step 1: Dissolve methyl 6-amino-2-benzoate (30g, 199mmol) in 200ml of methanol, add hydrazine hydrate solution (21g, 420mmol), and react at 80℃ for 8h. After the reaction is complete, cool the reaction solution to 0℃, filter, wash the obtained solid with methanol, and dry to obtain 25g of the target product, with a yield of 83%. 1 H NMR (400MHz, DMSO-d6) δ9.53(s,1H),7.13–6.98(m,2H),6.92(d,J=7.7Hz,1H),6.67(dd,J=7.9,1.6Hz,1H),5.23(s,2H),4.41(s,2H).

[0131] Step 2: 3-Aminobenzoylhydrazide (4 g, 26 mmol) and triethyl orthoformate were dissolved in 100 ml of 1,4-dioxane and reacted at 75 °C for 2 hours. Isopropylamine (1.24 g, 21 mmol) and acetic acid (5 g, 83 mmol) were added sequentially, and the reaction was carried out at 100 °C for 12 hours. After the starting materials had reacted completely, the reaction solution was concentrated, and the residue was subjected to silica gel column chromatography (methanol:dichloromethane = 1:30) to obtain 3.79 g of the target compound, with a yield of 82%.

[0132] Following the method in step 1.2 of Example 1, the target compound was prepared by acylation of intermediate 3-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)aniline with 1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid, with a yield of 21%. 1H NMR (400MHz, DMSO-d6) δ12.82(s,1H),8.62(d,J=8.0Hz,2H),8.37(d,J=15.1Hz,1H),7.81(d,J=8.0Hz,1H),7.68(d,J=7 .8Hz,1H),7.54(t,J=7.9Hz,1H),3.96(s,3H),3.65(ddd,J=10.9,7.3,3.9Hz,1H),1.12–1.03(m,2H),0.99–0.92(m,2H). 13 C NMR (126MHz, DMSO-d6)δ=163.93,159.73,152.79,151.43,149.48,146.14,145.63,142.47,140.13,135.91,13 1.32,130.89,124.77,123.78,119.35,115.84,114.43,112.71,110.55,62.94,33.71,28.64,12.56,7.63.ESI m / z:444.1396[M+H] + Found: 444.1416

[0133] Example 4

[0134] Synthesis of Compound 4

[0135] Preparation of N-(6-(4-cyclobutyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0136] Following the method in step 1.1 of Example 1, intermediate 3-(4-cyclobutyl-4H-1,2,4-triazol-3-yl)aniline was prepared by cyclization reaction with cyclobutylamine using intermediate 6-aminopyridinecarboxylhydrazine as a raw material, with a yield of 80%.

[0137] Following the method in step 1.2 of Example 1, the target compound was prepared by acylation of intermediate 3-(4-cyclobutyl-4H-1,2,4-triazol-3-yl)aniline with 1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid, with a yield of 16%. 1H NMR (400MHz, DMSO-d6) δ13.88(s,1H),12.81(s,1H),8.98(s,1H),8.67(s,1H),8.31(d,J=8.2Hz,1H),8.00(t,J=8.0Hz,1 H),7.83(d,J=7.6Hz,1H),5.41–5.24(m,1H),3.97(s,3H),3.17(s,2H),2.63(dd,J=16.4,7.9Hz,2H),1.94–1.72(m,2H). 13 C NMR (101MHz, DMSO-d6) δ = 162.37, 151.28, 150.71, 146.30, 144.63, 140.30, 136.02, 118. 94,116.28,114.20,102.73,50.92,49.01,36.20,31.11,14.88.ESIm / z:459.1505[M+H] + Found: 459.1525

[0138] Example 5

[0139] Synthesis of Compound 5

[0140] Preparation of N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0141] Following the method in step 1.1 of Example 1, the intermediate 6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine was prepared by cyclization reaction with cyclopentylamine using the intermediate 6-aminopyridinecarboxylhydrazine as the raw material, with a yield of 76%.

[0142] Following the method in step 1.2 of Example 1, the target compound was prepared by acylation of the intermediate 6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine with 1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid, with a yield of 37%. 1H NMR (400MHz, DMSO-d6) δ13.29(s,1H),8.88(s,1H),8.63(s,1H),8.35(d,J=8.2Hz,1H),8.01(t,J=8.0Hz,1H),7. 84(d,J=7.6Hz,1H),5.43(m,J=14.4,7.2Hz,1H),3.95(s,3H),2.35–2.22(m,2H),1.95–1.82(m,4H),1.74(s,2H). 13 C NMR (151MHz, DMSO-d6)δ=170.38,169.34,159.62,152.31,151.96,146.61,146.23,143.98,143 .34,140.24,138.41,131.21,114.33,113.57,111.95,108.91,57.97,34.27,33.71,23.84.ESI m / z:473.1661[M+H] + Found: 473.1684

[0143] Example 6

[0144] Synthesis of Compound 6

[0145] Preparation of N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(2-fluorobenzyl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0146] Following the method in step 1.2 of Example 1, the target compound was prepared by acylation of the intermediate 6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine with 7-(2-fluorobenzyl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid, with a yield of 65%. 1H NMR (400MHz, DMSO-d6) δ10.81(s,1H),8.82(s,1H),8.61(s,1H),8.27(d,J=8.3Hz,1 H),8.04(s,1H),7.86(d,J=7.5Hz,1H),7.69(ddd,J=23.2,7.6,6.1Hz,1H),7.38–7. 30(m,1H),7.23–7.16(m,1H),7.07(s,1H),5.71(s,2H),5.57–5.47(m,1H),4.14(s, 3H), 2.07 (dd, J=13.5, 5.4Hz, 2H), 1.81–1.67 (m, 4H), 1.59 (dd, J=10.1, 5.9Hz, 2H). 13 C NMR (126MHz, DMSO-d6)δ=164.47,163.75,161.66,161.34,160.47,159.70,151.37, 151.00,149.44,148.98,146.68,143.94,140.24,135.04,133.19,130.86,130.78,1 30.75,130.72,125.01,124.99,124.81,123.79,123.68,122.61,115.84,115.67,1 14.65,113.12,106.48,62.95,62.92,57.28,52.89,34.93,34.88,33.69,23.97.ESI m / z:581.2037[M+H] + Found: 581.2059

[0147] Example 7

[0148] Synthesis of Compound 7

[0149] Preparation of N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(4-methoxybenzyl)-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0150] Following the method in step 1.2 of Example 1, the target compound was prepared by acylation of the intermediate 6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine with 1-(4-methoxybenzyl)-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid, with a yield of 44%. 1H NMR (400MHz, DMSO) δ14.09(s,1H),12.58(s,1H),8.88(s,1H),8.72(s,1H),8.36(d,J=8.3Hz,1H),8.05(t,J=8.0Hz,1H),7.88(d,J=7.6Hz,1H),7.32 (d,J=8.6Hz,2H),6.94(d,J=8.6Hz,2H),5.55(s,2H),5.48–5.36(m,1H),3 .73(s,3H),2.36–2.23(m,2H),1.91(dd,J=12.5,5.7Hz,4H),1.74(s,2H). 13 C NMR (126MHz, DMSO) δ = 159.62, 151.15, 150.95, 146.70, 144.01, 140.45, 129.90, 12 7.74,119.56,116.87,114.62,114.35,102.88,57.99,55.60,51.59,33.73,23.89.

[0151] Example 8

[0152] Synthesis of Compound 8

[0153] 8.1 Preparation of 5-bromo-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid

[0154]

[0155] 5-Bromo-2-oxo-1,2-dihydropyridine-3-carboxylic acid (10 g, 31 mmol), 1-(bromomethyl)-2-fluorobenzene (17 g, 90 mmol), and potassium carbonate (12 g, 87 mmol) were dissolved in acetonitrile (300 mL) and reacted at 70 °C for 6 h. After the reaction was completed, the solution was concentrated, and a solid precipitated. The solid was filtered to give 12 g of the target product, with a yield of 79%. 1 H NMR (500MHz, DMSO-d6) δ13.97(s,1H),8.72(d,J=2.3Hz,1H),8.38(d,J=2.4Hz,1H),7.39(dd,J=13.7,6.9Hz,1H),7.32–7.11(m,3H),5.33(s,2H).

[0156] 8.2 Preparation of 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide

[0157]

[0158] 5-Bromo-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid (2 g, 6 mmol), 6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridine-2-amine (2.5 g, 12 mmol), and HATU (3.5 g, 9 mmol) were added to DMF (15 mL) and reacted at room temperature. Once the solvent became clear, DIPEA (1.6 g, 12 mmol) was added, and the reaction was continued at 40 °C for 4 h. After the reaction was complete, 30 mL of water was added, and a solid precipitated. The solid was filtered to obtain the crude product, dissolved in CH3OH, and subjected to silica gel column chromatography (DCM / CH3OH = 30:1) to give 2.3 g of the target compound in 74% yield. 1 H NMR (400MHz, DMSO-d6) δ12.40(s,1H),8.71–8.66(m,2H),8.51(d,J=2.8Hz,1H),8.29(d,J=8.2Hz,1H),8.02(t,J=8.0Hz,1H),7.86(d,J=7.6Hz,1H) ,7.38(s,1H),7.31(s,1H),7.25(d,J=10.0Hz,1H),7.20(d,J=7.6Hz,1H), 5.32(s,2H),3.76(s,1H),1.14–1.06(m,2H),1.03(dd,J=5.8,3.3Hz,2H).

[0159] 8.3 Preparation of 5-(1-cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide

[0160]

[0161] 5-Bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (200 mg, 0.39 mmol), 1-cyclopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhexacyclopentan-2-yl)-1H-pyrazole (200 mg, 0.7 mmol), bis(triphenylphosphine)palladium dichloride (88 mg, 0.1 mmol), and cesium carbonate (128 mg, 4 mmol) were dissolved in 1,4-dioxane and water, and the mixture was heated to 100 °C for 4 h under nitrogen protection. After the reaction was completed, the solution was concentrated, extracted with DCM (30 ml x 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was subjected to silica gel column chromatography (DCM / CH3OH = 30:1) to give 129 mg of the target compound, with a yield of 61%. 1 H NMR(400MHz, DMSO-d6)δ12.66(s,1H),8.75(d,J=2.7Hz,1H),8.69–8.65(m, 2H),8.37–8.29(m,2H),8.03(t,J=8.0Hz,1H),7.85(d,J=6.8Hz,2H),7.38(t d,J=7.4,1.6Hz,1H),7.26(dd,J=6.7,4.8Hz,2H),7.19(d,J=7.7Hz,1H),5.3 7(s,2H),3.77(dd,J=7.4,3.6Hz,2H),1.16–1.06(m,4H),1.05–0.95(m,4H). 13 C NMR (101MHz, DMSO-d6)δ=162.08,160.94,152.55,151.21,146.30,146.08,142.72,140.40,140.16,135.96,130.28,130.25,127.25,1 25.06,125.02,123.42,123.28,119.85,119.20,116.94,115.98,115.77,114.34,113.38,48.25,48.21,33.34,29.19,8.09,6.76.ESI m / z:537.2163[M+H] + Found: 537.2184

[0162] Example 9

[0163] Synthesis of Compound 9

[0164] Preparation of N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide)

[0165] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 33%. 1 H NMR (400MHz, DMSO-d6) δ12.68(s,1H),8.76(d,J=2.7Hz,1H),8.67(d,J=3.9Hz, 2H),8.37–8.30(m,2H),8.04(t,J=8.0Hz,1H),7.88–7.83(m,2H),7.38(td,J=7. 4,1.7Hz,1H),7.27(d,J=9.2Hz,2H),7.19(d,J=7.5Hz,1H),5.38(s,2H),4.52(s ,1H),3.77(s,1H),1.46(d,J=6.7Hz,6H),1.14–1.10(m,2H),1.06–1.01(m,2H). 1 3C NMR(101MHz, DMSO)δ=162.13,161.87,160.94,159.43,152.57,151.23,146.32,146.06,142.74,140.27,140.16,135.40,130.47,130.38,130 .26,130.22,125.05,123.46,123.32,119.85,119.22,116.74,115.98 ,115.77,114.35,113.69,53.74,48.20,29.49,29.17,23.13,8.08.ESI m / z: 539.2319 [M+H] + Found: 539.2331.

[0166] Example 10

[0167] Synthesis of Compound 10

[0168] Preparation of 5-(1-cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide

[0169] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 1-cyclobutyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 61%. 1 H NMR (400MHz, DMSO-d6) δ12.66(s,1H),8.76(d,J=2.6Hz,1H),8.68(s,2H),8.40–8.31(m,2H),8.03(t,J=8.0Hz,1H),7.93–7.82(m,2H),7.43–7.34 (m,1H),7.30–7.14(m,3H),5.37(s,2H),4.98–4.61(m,1H),3.89–3.63(m ,1H),2.50–2.36(m,4H),1.82(td,J=10.0,4.3Hz,2H),1.19–0.92(m,4H). 13 C NMR (151MHz, DMSO-d6)δ=162.12,160.93,152.55,151.21,146.30,146.06,142.73,140.27,140.16,135.39,130.44,130.39,13 0.23,130.21,125.04,123.43,119.83,119.21,116.73,115.94,115.80,114.34,113.68,53.73,48.22,29.18,23.14,8.08.ESI m / z:551.2319[M+H] + Found: 551.2345.

[0170] Example 11

[0171] Synthesis of Compound 11

[0172] Preparation of N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-6-oxo-1,6-dihydro-[3,3'-bipyridine]-5-carboxamide

[0173] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 3-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)pyridine via the Suzuki reaction, with a yield of 16%. 1 H NMR (500MHz, DMSO-d6) δ12.69 (s, 1H), 11.24 (s, 1H), 8.86 (d, J = 2.8Hz, 1H), 8.73–8. 65(m,2H),8.36(d,J=8.3Hz,1H),8.03(t,J=8.0Hz,1H),7.89–7.81(m,2H),7.52(d,J =8.4Hz,1H),7.42(t,J=2.7Hz,1H),7.40–7.31(m,3H),7.22(dt,J=15.0,8.6Hz,2H) ,6.52(s,1H),5.46(s,2H),3.84–3.74(m,1H),1.18–1.09(m,2H),1.07–1.00(m,2H). 13 C NMR (151MHz, DMSO-d6)δ=162.25,161.08,152.57,151.24,146.30,146.06,143.63,141.80,140.15,135.96,130.66,130.63,130.50,130.44, 128.77,126.98,126.26,125.03,123.44,121.80,119.72,119.45,119 .20,117.86,115.96,115.82,114.38,112.64,101.95,29.20,8.10.ESI m / z: 546.2054 [M+H] + Found: 546.2071.

[0174] Example 12

[0175] Synthesis of Compound 12

[0176] Preparation of N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0177] 12.1 Preparation of 2-(3-(6-aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)prop-1-ol

[0178]

[0179] Step 1: 50 g of methyl 6-aminopyridinecarboxylate (329 mmol) was dissolved in 200 ml of methanol, and 32 g of hydrazine hydrate solution (420 mmol) was added. The mixture was reacted at 80 °C for 8 h. After the reaction was completed, the reaction solution was cooled to 0 °C, filtered, and the resulting solid was washed with methanol and dried to obtain 42 g of the target product, with a yield of 84%.

[0180] Step 2: 6-Aminopyridine carboxyhydrazide (5 g, 26 mmol) and triethyl orthoformate were dissolved in 100 ml of 1,4-dioxane and reacted at 75 °C for 2 hours. Then, 2-aminoprop-1-ol (2.5 g, 33 mmol) and acetic acid (5 g, 83 mmol) were added sequentially, and the reaction was carried out at 100 °C for 12 hours. After the starting materials had reacted completely, the reaction solution was concentrated, and the residue was subjected to silica gel column chromatography (methanol:dichloromethane = 1:30) to obtain 5.62 g of the target compound, with a yield of 78%. 1 H NMR (400MHz, DMSO-d6) δ8.69(s,1H),7.52(dd,J=8.3,7.3Hz,1H),7.18(dd,J=7.4,0.9Hz,1H),6.52(dd,J =8.3,0.9Hz,1H),6.17(s,1H),5.50–5.28(m,1H),5.03(s,1H),3.72–3.51(m,1H),1.43(d,J=6.9Hz,2H).

[0181] 12.2 Preparation of N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide

[0182]

[0183] 1-Methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid (500 mg, 1.92 mmol) was dissolved in 10 mL of sulfoxide and reacted at 40 °C for 2 h. The solvent was concentrated, and 2-(3-(6-aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)prop-1-ol (420 mg, 2 mmol) was added and dissolved in dichloromethane (15 mL). The reaction was carried out at room temperature for 5 h. After the reaction was completed, the reaction solution was concentrated, and the residue was subjected to column chromatography (CH3OH / DCM = 1:30) to give 100 mg of the target compound, with a yield of 11%. 1 H NMR (400MHz, DMSO) δ15.25(s,1H),8.79(s,1H),8.46(s,1H),8.36(d,J=8.0Hz,1H),7.96(t,J=8.0Hz,1H),7.74(d ,J=7.5Hz,1H),5.33(d,J=5.2Hz,1H),5.12(s,1H),3.80(s,3H),3.73(s,1H),1.89(s,2H),1.54(d,J=6.9Hz,3H). 13 C NMR(101MHz,DMSO)δ=170.57,165.83,154.30,152.42,151.15,146.65,144.21 ,139.88,131.30,118.41,114.96,114.20,64.66,54.51,33.71,29.28,18.08.

[0184] Example 13

[0185] Synthesis of Compound 13

[0186] Preparation of 5-(1-cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide

[0187]

[0188] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 1-cyclopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 57%. 1H NMR(600MHz,DMSO-d6)δ13.01(d,J=6.4Hz,1H),12.90(s,1H),8.69(d,J=2.7Hz,2 H),8.36(d,J=8.2Hz,1H),8.30(s,1H),8.11(dd,J=6.4,2.8Hz,1H),8.04(t,J=8. 0Hz,1H),7.90–7.85(m,2H),3.82(tt,J=7.6,4.1Hz,1H),3.73(tt,J=7.4,3.8Hz, 1H),1.17(td,J=7.4,5.5Hz,2H),1.11–1.04(m,4H),0.98(td,J=7.4,4.9Hz,2H). 13 C NMR(151MHz,DMSO)δ=162.40,162.05,152.49,151.33,146.28,146.11,142.99,140.15,136.25,136.08,127.06,11 9.78,118.94,117.11,114.29,113.45,40.40,40.26,40.12,39.98,39.84,39.70,39.57,33.30,29.40,8.20,6.76.

[0189] Example 14

[0190] Synthesis of Compound 14

[0191] Preparation of N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide)

[0192] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 54%. 1H NMR(600MHz,DMSO-d6)δ13.00(d,J=6.3Hz,1H),12.91(s,1H),8.74–8.64(m,2H ),8.36(d,J=8.3Hz,1H),8.30(s,1H),8.12(dd,J=6.4,2.8Hz,1H),8.04(t,J=8 .0Hz,1H),7.88(t,J=3.8Hz,2H),4.49(hept,J=6.7Hz,1H),3.82(tt,J=7.6,4. 1Hz,1H),1.45(d,J=6.7Hz,5H),1.17(td,J=7.4,5.5Hz,2H),1.09–1.03(m,2H). 13 C NMR (151MHz, DMSO) δ = 162.43, 162.04, 152.51, 151.34, 146.31, 146.11, 142.99, 140.15, 136.12, 135.48, 124.90, 119.76, 1 18.94,116.92,114.29,113.76,53.66,40.40,40.26,40.12,39.98,39.84,39.71,39.57,30.62,29.89,29.38,23.15,8.19.

[0193] Example 15

[0194] Synthesis of Compound 15

[0195] Preparation of 5-(1-cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide

[0196] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 1-cyclobutyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 16%. 1H NMR(600MHz,DMSO-d6)δ13.04–12.99(m,1H),12.91(d,J=5.6Hz,1H),8.73–8.64(m,2H),8.36(d ,J=8.0Hz,2H),8.16–8.11(m,1H),8.04(t,J=8.0Hz,1H),7.91(s,1H),7.88(d,J=7.6Hz,1H),4. 83(p,J=8.4Hz,1H),3.82(tt,J=7.6,4.1Hz,1H),2.47(td,J=9.5,2.7Hz,1H),2.40(qt,J=7.7,2 .9Hz,2H),1.80(ddt,J=14.7,13.0,5.5Hz,2H),1.17(td,J=7.4,5.5Hz,2H),1.11–1.02(m,2H).

[0197] Example 16

[0198] Synthesis of Compound 16

[0199] Preparation of 6-(1-cyclopropyl-1H-pyrazol-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-3-carboxamide

[0200]

[0201] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 6-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-3-carboxamide with 1-cyclopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 26%. 1 H NMR (600MHz, DMSO-d6) δ11.12(s,1H),9.26(d,J=2.2Hz,1H),8.85(d,J=2.2Hz,1 H),8.67(s,1H),8.47(s,1H),8.34–8.31(m,2H),8.17(dd,J=8.8,2.0Hz,1H),8. 11–8.05(m,3H),7.86(dd,J=7.6,0.9Hz,1H),4.30(tt,J=7.5,4.1Hz,1H),3.80( tt,J=7.5,3.8Hz,1H),1.14–1.10(m,2H),1.06–1.01(m,4H),0.97–0.93(m,2H). 13C NMR (151MHz, DMSO) δ = 165.83, 152.90, 151.85, 148.72, 147.96, 146.14, 145.41, 139.95, 137.09, 136.59, 132 .18,130.31,129.74,128.35,128.13,127.39,123.72,121.14,119.54,115.34,33.40,28.81,7.65,6.82.ESI m / z:463.1995[M+H] + ; 463.1990.

[0202] Example 17

[0203] Synthesis of Compound 17

[0204] Preparation of 6-(1-cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-3-carboxamide

[0205] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 6-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-3-carboxamide with 1-cyclobutyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 37%. 1 H NMR (600MHz, DMSO-d6) δ11.12(s,1H),9.26(d,J=2.3Hz,1H),8.86(d,J=2.2Hz,1H),8.67(s,1H), 8.51(d,J=0.8Hz,1H),8.33(dd,J=8.3,1.1Hz,2H),8.18(dd,J=8.8,2.0Hz,1H),8.13–8.05(m,3H ),7.86(dd,J=7.6,0.8Hz,1H),4.94–4.83(m,1H),4.30(tt,J=7.6,4.1Hz,1H),2.54(dd,J=9.8,2 .6Hz,1H),2.47–2.40(m,2H),1.91–1.80(m,2H),1.04(td,J=7.4,5.4Hz,2H),0.97–0.91(m,2H). 13C NMR (151MHz, DMSO) δ = 165.84, 152.90, 151.85, 148.71, 147.96, 146.15, 145.41, 139.96, 137.07, 136.60, 132.32, 130.33, 129.75, 128.14, 127.41,127.00,123.71,121.22,119.55,115.34,55.44,40.40,40.26,40.12,39.98,39.84,39.70,39.57,30.59,28.81,14.61,7.65.ESI m / z:477.2151[M+H] + ; 477.2148.

[0206] Example 18

[0207] Synthesis of Compound 18

[0208] Preparation of N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1-isopropyl-1H-pyrazol-4-yl)quinoline-3-carboxamide

[0209] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 6-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-3-carboxamide with 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 22%. 1 H NMR (600MHz, DMSO-d6) δ11.11(s,1H),9.26(d,J=2.2Hz,1H),8.86(d,J=2.2Hz,1H),8.67 (s,1H),8.46(s,1H),8.33(dd,J=5.2,3.2Hz,2H),8.18(dd,J=8.7,2.0Hz,1H),8.12–8.04 (m,3H),7.86(dd,J=7.6,0.8Hz,1H),4.56(hept,J=6.7Hz,1H),4.30(tt,J=7.5,4.1Hz,1 H), 1.49 (d, J=6.6Hz, 5H), 1.04 (td, J=7.4, 5.2Hz, 2H), 0.96 (ddd, J=7.3, 5.6, 4.3Hz, 2H). 13C NMR (151MHz, DMSO) δ = 165.85, 152.90, 151.85, 148.64, 147.92, 146.15, 145.41, 139.95, 136.59, 136.58, 132.49, 130.32, 129.73, 128.13, 127.4 3,126.12,123.57,120.91,119.54,115.34,53.77,40.40,40.26,40.12, 39.98,39.85,39.71,39.57,28.81,23.17,7.65.ESIm / z:465.2151[M+H] + ; 465.2145.

[0210] Example 19

[0211] Synthesis of Compound 19

[0212] Preparation of N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(6-methylpyridin-3-yl)quinoline-3-carboxamide

[0213] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 6-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-3-carboxamide with 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxoborane-2-yl)pyridine via the Suzuki reaction, with a yield of 24%. 1 H NMR (400MHz, DMSO) δ11.18(s,1H),9.37(d,J=2.2Hz,1H),9.03(d,J=2.0Hz,1H),8.97(d,J= 2.2Hz,1H),8.68(s,1H),8.54(d,J=1.9Hz,1H),8.35–8.32(m,1H),8.30–8.26(m,1H),8.22 (d,J=8.8Hz,1H),8.18(dd,J=8.1,2.5Hz,1H),8.13–8.05(m,1H),7.87(dd,J=7.6,0.7Hz,1 H),7.45(d,J=8.1Hz,1H),4.31(s,1H),2.56(s,3H),1.07–1.01(m,2H),1.00–0.92(m,2H).

[0214] Example 20

[0215] Synthesis of Compound 20

[0216] Preparation of N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazol-4-yl)-1H-indazole-3-carboxamide

[0217]

[0218] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indazole-3-carboxamide with 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 13%. 1 H NMR(500MHz,DMSO-d6)δ13.26(s,1H),10.86(s,1H),8.68(d,J=6.7Hz,2H),8.48(s,2H) ,8.32(d,J=8.3Hz,1H),8.10(s,1H),8.04(t,J=8.0Hz,1H),7.98(dd,J=8.7,1.6Hz,1H), 7.81(d,J=7.6Hz,1H),7.64(d,J=8.8Hz,1H),4.62(p,J=6.7Hz,1H),4.32(tt,J=7.4,4. 1Hz,1H),1.50(d,J=6.6Hz,6H),1.06–0.99(m,2H),0.97(dq,J=7.5,4.5,3.9Hz,2H).ESI m / z:454.2104[M+H] + :454.2133.

[0219] Example 21

[0220] Synthesis of Compound 21

[0221] Preparation of 5-(1-cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indazole-3-carboxamide

[0222] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indazole-3-carboxamide with 1-cyclobutyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 32%. 1H NMR(600MHz,DMSO-d6)δ13.27(s,1H),10.86(s,1H),8.72–8.65(m,2H),8.55(s,1H),8.32(dd, J=8.4,0.9Hz,1H),8.13(s,1H),8.05(t,J=8.0Hz,1H),7.98(dd,J=8.7,1.5Hz,1H),7.82(dd,J =7.6,0.9Hz,1H),7.65(d,J=8.7Hz,1H),5.00–4.89(m,1H),4.31(tt,J=7.5,4.1Hz,1H),2.69– 2.51(m,4H),2.47–2.37(m,2H),1.90–1.76(m,2H),1.11–0.91(m,4H).ESIm / z:466.2104[M+H] + :466.2124.

[0223] Example 22

[0224] Synthesis of Compound 22

[0225] Preparation of 5-(1-cyclopropyl-1H-pyrazol-4-yl)-1-(2-fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide)

[0226]

[0227] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-1-(2-fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 1-cyclopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 36%. 1H NMR (400MHz, DMSO-d6) δ12.66(s,1H),8.33(d,J=8.7Hz,1H),8.04(t,J=8.0Hz,1H),7.38(t,J=7.0Hz,1H),7.34–7.22(m,1H),5.37(s,1H),5.20(q, J=6.1Hz,1H),5.02(s,1H),3.76(tt,J=7.3,3.8Hz,1H),3.72(d,J=5.5Hz, 1H), 1.49 (d, J=6.9Hz, 1H), 1.08 (m, J=3.3, 2.9Hz, 1H), 1.05–0.94 (m, 1H).

[0228] Example 23

[0229] Synthesis of Compound 23

[0230] Preparation of 1-(2-fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide

[0231] Following the method in step 8.3 of Example 8, the target compound was prepared by reacting the intermediate 5-bromo-1-(2-fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide with 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentan-2-yl)-1H-pyrazole via the Suzuki reaction, with a yield of 45%. 1 H NMR (400MHz, DMSO-d6) δ12.68(s,1H),8.80(s,1H),8.75(d,J=2.7Hz,1H),8.68(d,J=2.7H z,1H),8.37–8.28(m,2H),8.04(t,J=8.0Hz,1H),7.87(d,J=0.8Hz,1H),7.81(dd,J=7.7,0 .9Hz,1H),7.45–7.33(m,1H),7.34–7.10(m,3H),5.38(s,2H),5.20(h,J=6.6Hz,1H),5.02 (t,J=5.4Hz,1H),4.52(h,J=6.6Hz,1H),3.77–3.66(m,2H),1.47(dd,J=12.8,6.8Hz,9H).

[0232] Biological Examples

[0233] The following biological experiments further illustrate the beneficial effects of the compounds of the present invention, but this should not be construed as the compounds of the present invention having only the following beneficial effects.

[0234] Example 24:

[0235] In vitro enzymatic activity of the compounds of this invention

[0236] Test samples: The chemical structural formulas of the compounds synthesized in the embodiments of the present invention are shown in Table 1, and the experimental materials are shown in Table 2.

[0237] Table 2 Experimental Materials

[0238]

[0239] Experimental methods:

[0240] 1. Compound dilution

[0241] 1) Dilute the compound of the present invention to 8 mM using DMSO.

[0242] 2) Dilute the above-mentioned compound of the present invention to 500 μM or 1 mM. Dilute the compound of the present invention in a two-fold serial dilution to 12 concentrations, with the highest concentration being 5 μM or 10 μM.

[0243] 3) Use a pipette to transfer 1 μL of the compound of the present invention into each well of a 384-well plate.

[0244] 2. Preparation of the reaction system

[0245] 1) Prepare the enzyme reaction system according to Table 3. This system contains kinases and the factors required for the reaction.

[0246] Table 3 Enzyme Reaction System

[0247]

[0248] 2) Add 2 μL of the enzyme system to each well.

[0249] 3) Add 2 μL of 2.5×ATP / Substrate Mix to each reaction well (see Table 4);

[0250] Table 4. 2.5×ATP / Substrate Mix System

[0251]

[0252] 4) After reacting the system at 37°C for 60 min, add 5 μL of ADP-Glo TM Reagent, incubate at 37°C for 40 min;

[0253] 5) Add 10 μL Kinase Detection Reagent, incubate at 37°C for 30 min, and detect using a multi-functional microplate reader.

[0254] 3. Data Analysis:

[0255] 3.1 Calculate using the following formula:

[0256] Residual enzyme rate % = (Average OD value of test group / OD value of blank group) × 100%

[0257] Enzyme inhibition rate = (1 - average OD value of test group / OD value of blank group) × 100%

[0258] IC was calculated using the regression-Probit method with SPSS statistical analysis software. 50 value.

[0259] 3.2 The results are as follows:

[0260] Table 5. Inhibitory activity of compounds against ASK1

[0261] Serial Number <![CDATA[ASK1 IC 50 (μM)]]> Serial Number <![CDATA[ASK1 IC 50 (μM)]]> Compound 1 0.49 Compound 14 0.004 Compound 2 2.66 Compound 15 0.002 Compound 3 2.70 Compound 16 0.519 Compound 4 0.98 Compound 17 0.13 Compound 5 2.18 Compound 18 0.068 Compound 6 1.02 Compound 19 1.22 Compound 7 0.708 Compound 20 0.161 Compound 8 0.002 Compound 21 0.50 Compound 9 0.009 Compound 22 0.007 Compound 10 0.28 Compound 23 0.011 Compound 11 2.37 GS-444217 0.007 Compound 12 0.179 MP7 >5 Compound 13 0.005

[0262] Table 6. Inhibitory activity of compounds against PDK1

[0263] Serial Number PDK1 inhibition@10μM (%) Serial Number PDK1 inhibition@10μM (%) Compound 1 27.45 Compound 14 22.11 Compound 2 42.85 Compound 15 19.78 Compound 3 27.96 Compound 16 26.03 Compound 4 31.94 Compound 17 18.54 Compound 5 37.72 Compound 18 27.72 Compound 6 29.90 Compound 19 24.35 Compound 7 14.20 Compound 20 46.11 Compound 8 13.63 Compound 21 39.19 Compound 9 23.80 Compound 22 12.99 Compound 10 40.27 Compound 23 17.49 Compound 11 20.99 GS-444217 0.04 Compound 12 44.38 MP7 65.09 Compound 13 32.19

[0264] The experimental results show that the compounds prepared in this invention can effectively inhibit the activities of both ASK1 and PDK1, exhibiting significant dual-target inhibition. In contrast, the positive control drug GS-444217 only inhibits ASK1 and has no inhibitory effect on PDK1. MP7 only inhibits PDK1 and has no inhibitory effect on ASK1.

[0265] Example 25

[0266] In vitro cellular inhibitory activity of the compounds of this invention

[0267] Test compound:

[0268] 1) Test sample: The preferred compound of this invention, the chemical structure of which is shown in Table 1

[0269] 2) Positive test results: GS-444217, MP7

[0270] Experimental materials:

[0271] 1) Cell lines: human embryonic lung fibroblasts (HFL-1), rat kidney fibroblasts (NRK-49F), and rat liver morphocytes (HSC-T6).

[0272] 2) Reagents and instruments: 96-well plates (Corning); fetal bovine serum (Gibco), F12K medium, DMEM medium (BI); biosafety cabinet, carbon dioxide incubator (ESCO); multi-functional microplate reader (Bio-tek).

[0273] Experimental methods:

[0274] 1) Cell culture: Cells in the logarithmic growth phase with a viable cell ratio of more than 90% were used in the experiments.

[0275] 2) Cell growth assay: Digest cells and dilute them to a concentration of 3-5 × 10⁻⁵. 4 Add 100 μL of cell suspension (3-5 × 10⁶ cells / mL) to each of the middle 60 wells of a 96-well plate. 3 100 μL of PBS was added to each of the 36 wells around the perimeter of the plate. The 96-well plate was incubated at 37°C with 5% CO2 until it adhered to the walls (entered the logarithmic growth phase). After removing the culture medium from the middle 60 wells, 100 μL of culture medium containing different concentrations of the compound was added to each well, with 3 replicates per group. The 96-well plate was incubated for 48 h. The culture medium containing the compound and the PBS were discarded. 100 μL of basal culture medium containing 10% CCK-8 was added to each well of the 96-well plate. The 96-well plate was incubated for 1-4 h and the OD value was measured at 450 nm using a microplate reader.

[0276] Data Analysis:

[0277] Calculate the cell proliferation rate and inhibition rate for each group.

[0278] Cell viability % = (Average OD value of test group / OD value of blank group) × 100%

[0279] Inhibition rate = (1 - average OD value of test group / OD value of blank group) × 100%

[0280] Calculate the half-maximal inhibitory concentration (IC50) of the drug. 50 Based on the drug's effective concentration and its growth inhibition rate on cells, the IC50 was calculated using the regression-Probit method with SPSS statistical analysis software. 50 value.

[0281] The experimental results are shown in Table 7:

[0282] Table 7 Growth inhibition rate of the compounds of the present invention on cells

[0283]

[0284] The experimental results above show that the compound prepared by the present invention has better inhibitory activity against lung, kidney and liver fibroblasts compared with the control drug, and is significantly better than the positive control drug. Therefore, the compound represented by general formula (I) or (II) provided by the present invention is an effective ASK1 / PDK1 inhibitor.

[0285] Example 26

[0286] Effects of the compounds of this invention on the ASK1 / PDK1 pathway

[0287] Test compound:

[0288] 3) Test samples: Compounds 8 and 9 are preferred in this invention, and their chemical structures are shown in Table 1.

[0289] Experimental materials:

[0290] 1) Cell line: Human embryonic lung fibroblasts (HFL-1)

[0291] 2) Reagents and instruments: 6-well plates (Corning); fetal bovine serum (Gibco); F12K medium; biosafety cabinet; carbon dioxide incubator (ESCO); multi-functional microplate reader (Bio-tek); chemiluminescence imaging system (Bio-Rad); constant voltage and constant current electrophoresis apparatus (Bio-Rad).

[0292] Experimental methods:

[0293] 1) Prepare 8% separating and stacking gels according to the gel concentration and volume. Add electrophoresis buffer (enough to completely submerge the stacking gel), and load the samples at a rate of 30 μg / lane. Add 5 μL of marker. Set the electrophoresis voltage for the stacking gel to 80V. Once the samples have uniformly migrated to the separating gel, adjust the voltage to 120V and continue electrophoresis. Stop the electrophoresis when the bromophenol blue is about to exit the gel.

[0294] 2) Prepare the transfer filter paper. Place the filter paper in the following order: sponge pad, two layers of filter paper, gel, PVDF membrane, two layers of filter paper, sponge pad. Place the gel at the negative electrode and the PVDF membrane at the positive electrode. Transfer at constant current for 2 hours.

[0295] 3) Wash the membrane 3 times with TBST for 5-10 minutes each time, then transfer it into TBST containing 5% skim milk powder for sealing. Shake at room temperature for 1-2 hours, and then blot dry the sealing solution with filter paper.

[0296] 4) Add primary antibody: Dilute mouse anti-human actin monoclonal antibody with primary antibody dilution buffer to a dilution of 1:5000 and incubate overnight at 4°C.

[0297] 5) Add secondary antibody: Dilute goat anti-rabbit HRP-lgG with TBST to a dilution of 1:5000, and shake at room temperature for 1 hour.

[0298] 6) Prepare the chemiluminescent solution in a 1:1 ratio: take 0.1 mL of reagent A and 0.1 mL of reagent B. Place the membrane protein side down in the mixed chemiluminescent solution for full contact and expose for 1–3 min.

[0299] Experimental results are as follows Figure 1 and Figure 2 As shown, PDK1 can phosphorylate its downstream factor AKT, thereby regulating cell proliferation, apoptosis, and cell differentiation. Experimental results showed that when HFL-1 cells were stimulated by TGF-β1, the phosphorylation levels of PDK1 and AKT proteins increased. After 24 hours of treatment with compounds 8 and 9, the phosphorylation levels of PDK1 and AKT were significantly inhibited, effectively regulating the PDK1 / AKT pathway. This experiment further verified that compound 8 did indeed have an inhibitory effect on PDK1 enzyme at a 13% inhibition rate at 10 μM.

[0300] like Figure 2 As shown, the effects of compounds 8 and 9 on the ASK1 pathway were examined. Results indicated that when HFL-1 cells were stimulated by TGF-β1, the phosphorylation levels of ASK1, P38, and JNK proteins increased. After 24 hours of treatment with compounds 8 and 9, the phosphorylation levels of ASK1, P38, and JNK were significantly inhibited, effectively regulating the ASK1 / P38 / JNK pathway. Western blotting experiments confirmed that the positive control drug GS-444217 had no inhibitory effect on PDK1 and AKT phosphorylation, and MP7 had no significant inhibitory effect on ASK1, P38, and JNK. However, compounds 8 and 9 simultaneously inhibited the ASK1 / PDK1 dual pathway, a result consistent with enzyme assays.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, ； in, R1 is , R12 is selected from C3-C8 cycloalkyl, methyl, ethyl, C3-C8 straight-chain or branched alkyl; R13 is selected from methyl, ethyl or propyl. R2 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R12 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, isopropyl, n-propyl, n-butyl, and isobutyl.

3. Compounds of formula (II) or pharmaceutically acceptable salts thereof: ； in, R3 is R14 is selected from C3-C8 cycloalkyl, methyl, ethyl, C3-C8 straight-chain or branched alkyl groups; R4 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

4. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein, R14 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, isopropyl, n-propyl, n-butyl, and isobutyl.

5. Compounds of general formula (III) or pharmaceutically acceptable salts thereof: ； in, X is C or N; R5 is hydrogen, C1-C3 alkyl, or halo-C1-C3 alkyl; R6 is hydrogen, C1-C3 alkyl, or halo-C1-C3 alkyl. R7 is hydrogen; R8 is C3-C6 cycloalkyl, C1-C6 alkyl, or C1-C6 hydroxyalkyl.

6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein, R6 is a methyl group.

7. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein, R8 is a C3-C6 cycloalkyl, C1-C4 alkyl, or C1-C4 hydroxyalkyl.

8. Compounds of formula (Ⅳ) or pharmaceutically acceptable salts thereof: ； in, Y is either C or N; R9 is pyrrole, substituted pyrrole, pyrazole, substituted pyrazole, imidazole, substituted imidazole, pyridine, substituted pyridine, pyrimidine, or substituted pyrimidine; the substituents on the substituted pyrrole, substituted pyrazole, substituted imidazole, substituted pyridine, and substituted pyrimidine are selected from C3-C6 cycloalkyl, C1-C4 alkyl, and C1-C4 haloalkyl groups. R10 is hydrogen; R11 is a C3-C6 cycloalkyl, C1-C6 alkyl, or C1-C6 hydroxyalkyl.

9. The compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein, The substituents on the substituted pyrrole, substituted pyrazole, substituted imidazole, substituted pyridine, and substituted pyrimidine are selected from isopropyl, n-propyl, n-butyl, and isobutyl.

10. The compound according to claim 8 or 9, or a pharmaceutically acceptable salt thereof, wherein, R9 is , , .

11. The compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein, R 11 It is C3-C6 cycloalkyl, C1-C4 alkyl, or C1-C4 hydroxyalkyl.

12. The following compounds or pharmaceutically acceptable salts thereof, wherein the compounds are selected from: N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 01) N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (compound 02) N-(3-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazole[3,4-b]pyridine-5-carboxamide (Compound 03) N-(6-(4-cyclobutyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (compound 04) N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 05) N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(2-fluorobenzyl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 06) N-(6-(4-cyclopentyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(4-methoxybenzyl)-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (Compound 07) 5-(1-Cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-Cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 08) N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 09) 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 10) N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-(2-fluorobenzyl)-6-oxo-1,6-dihydro-[3,3'-bipyridine]-5-carboxamide (compound 11) N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-6-oxo-3-(trifluoromethyl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (compound 12) N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (compound 13) 5-(1-Cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-Cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 14) 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 15) 6-(1-Cyclopropyl-1H-pyrazole-4-yl)-N-(6-(4-Cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)quinoline-4-carboxamide (Compound 16) 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indazole-3-carboxamide (Compound 17) N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1-isopropyl-1H-pyrazol-4-yl)quinoline-4-carboxamide (Compound 18) N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(6-methylpyridin-3-yl)quinoline-3-carboxamide (Compound 19) N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazole-4-yl)-1H-indazole-3-carboxamide (Compound 20) 5-(1-Cyclobutyl-1H-pyrazole-4-yl)-N-(6-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indazole-3-carboxamide (Compound 21) 5-(1-Cyclopropyl-1H-pyrazole-4-yl)-1-(2-fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (Compound 22) 1-(2-Fluorobenzyl)-N-(6-(4-(1-hydroxypropyl-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1-isopropyl-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carboxamide (compound 23).

13. The method for synthesizing the compound according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Intermediate 1 is synthesized from an aromatic heterocyclic carboxylic acid via an acylation reaction to obtain intermediate 2; Step 2: Intermediate 2 reacts with aromatic heterocyclic borate esters via the Suzuki reaction to yield compound of general formula (Ⅰ); The synthesis route is as follows: 。 14. Use of the compound according to any one of claims 1-12 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and / or prevention of diseases associated with the dual targets of ASK1 / PDK1.

15. The application as described in claim 14, characterized in that, The ASK1 / PDK1 dual-target disease is fibrosis.

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